seminario biologia molecular hd
DESCRIPTION
protrombina, apo A-IV y Haptoglobina incrementadas en la enfermedad de huntingtonTRANSCRIPT
“INCREASED
PROTHROMBIN, APOLIPOPROT
EIN A-IV, AND HAPTOGLOBIN IN
THE CEREBROESPIAL FLUID OF
PATIENTS WITH HUNTINGTON´S
DISEASE.”Yen-Chu Huang.
Maria Camila Montufar Pantoja
Heyly Milena Ortega
III SEMESTER
2011
UNIVERSIDAD PONTIFICIA BOLIVARIANA
GENERAL OBJETIVE
Demonstrate how Prothrombin,
Apolipoprotein A-IV and Haptoglobin
concentrations in CSF has an important
role in the HD evolution and
compare these concentrations in HD and
control patients .
INTRODUCTION
HD is a hereditary, degenerative brain disorder. It
is caused by an unstable CAG trinucleotide.
Today there are others proteins involved in HD
development and symptoms (Apolopoprotein A-
IV, Haptoglobin , Prothrombin) and its important to
clarify through proteomics studies in CSF wich
are the concentrations (up or down) and if this
levels of biomarkers are correlated in HD.
PROTHROMBIN
Plasma protein synthesized
in the liver.
It plays an important function in
coagulation process by
reaction with the thromboplastin
enzyme , with Ca ++ cation and
V activate factor as coenzymes.
In HD prothrombin
proinflammatory agent : DISEASE
STATUS BIOMARKER
Tromboplastina
Ca++
Xa
Va
APOLIPOPROTEIN AIV (APO
AIV)
glycoprotein synthesized by the human intestine and
liver
The formation of chylomicrons acts as a signal for the
induction of Apo AIV synthesis
alters the activity of the enzymes (LPL and LCAT) of
lipoprotein metabolism and cholesterol efflux from
extrahepatic tissues
Is involved in the long-term regulation of both food
intake and body weight
HAPTOGLOBIN
protein produced by the liver
haptoglobin binds free hemoglobin (Hb) released from
erythrocytes with high affinity and thereby inhibits its
oxidative activity
The haptoglobin-hemoglobin complex will then be
removed by the reticuloendothelial system
It prevents loss of iron through the kidneys and
protecting the kidneys from damage by hemoglobin
ENFERMEDAD DE HUNITNGTON
- HD
Progressive neurodegener
ative
disorder with autosomal
dominant inheritance:
- Movement Disorders.
- Cognitive impairment.
- Psychiatric disorders.
MATERIALES Y
METODOS
LCR y suero extraido de 9 HD ( 5 hombres- 4
mujeres).
LCR y suero de 18 pacientes tipo control (7
hombres- 11 mujeres).
Cuatro grupos:
- 1 Electroforesis de dos dimensiones.
- 3 Cuantificacion de protrombina, Apo-A IV y
haptoglobina.
PREPARACION DE LAS
MUESTRAS
Muestras de LCR• Centrifugados.
• Almacenados a -80°
Muestras de Suero
• Almacenados a 4°c durante 1 hora.
• Centrifugados.
• Almacenados a -80°c.
ELECTROFORESIS DE DOS
DIMENSIONES (2-DE )
Técnica de alta resolución cuyo
objetivo es la separación de mezclas
de proteínas altamente complejas.
las proteínas son separadas por dos
criterios físicos.
- Punto isoeléctrico
(isoelectroenfoque).
- Masa molecular
por electroforesis discontinua en gel
de poliacrilamida en presencia de
SDS (SDS-PAGE).
DIGESTION EL GEL
Forma parte de la preparación de las muestras para
la espectrometría de masas, consta de cuatro
pasos:
- Decoloración.
- Reducción y alquilación.
- Corte de la proteína serina proteasa tripsina
- Extracción del péptido generado.
ESPECTROMETRIA DE MASA
técnica experimental que permite la medición de iones derivados de
moléculas
se basa en el fenómeno conocido como desbastado (sputtering,
en inglés) de partículas centradas en un blanco, que son
bombardeadas por iones, átomos o moléculas. Dependiendo del
intervalo de energía de la partícula primaria, ocurren colisiones.
el patrón de fragmentación resultante así como los
iones residuales constituyen el espectro de masas
WESTERN BLOT
Busco proteínas
especificas
Uso ac
Uso técnica SDS-
PAGE
Separación de
proteínas por
electroforesis en gel
ELISA
Ensayo por inmunoabsorción ligado a enzimas
Fosfatasa alcalina—peroxidasa de rabano
Detección ag-ac
3 tipos:
Ensayo tipo sandwich
Ensayo competitivo
Ensayo indirecto
RESULTADOS
Representative Western blots of prothrombin (72 kD)and albumin (64 kD) in CSF and serum of HD patientsand their corresponding controls.
Representative Western blots of Apo A-IV (46 kD) and
albumin (64 kD) in CSF and serum, respectively, of HD
patients and their corresponding controls (C).
RESULTADOS
Qalb, CSF haptoglobin, and serum haptoglobin
concentration in HD patients.
RESULTADOS
DISCUSSION
AUTOR OPINION YES NO
Bjorkqvist M.
The mutant huntingtin
may enchance the ability
of microglia to produce
proinflamatory mediators
(IL-6, IL-8 and TNF-a)
and contribute to
neurodegeneration in
HD.
Djoussé L.
In HD, weigth loss is a
characteristic feature in
advanced as well early
stage.
Fang Q, Strand In proteome
analyses several the
neuronal proteins
were found to be
reduced
Underwood BR A serum metabolic
profile indicative of a
pro-catabolic
phenotype in early
HD
CONCLUTIONS
The proteomic studies in CFS in patients HD andcontrol patients are very important to determinateif the increase levels of Haptoglobin,Apolipoprotein A-IV and Prothrombin arecorrelated with HD development.
It is important to recognize this proteins likespecifics biomarkers in HD; thats why theconcentrations of each one were quantified inCSF and serum; thus indirectly evaluate the BBBintegrity.
CONCLUTIONS
To be able to correlate the levels of these proteins with other mental
diseases such as Alzheimer's, multiple sclerosis, among others; allow
us to determine whether the activity of proteins mentioned in the study
are a common phenomenon in a variety of neurodegenerative-tive
disease or are specifically for HD.
Becouse of the intact values of BBB and the increase levels of proteins,
it suggest that they are produced in the CNS itself and that the relation
within de concentrations of proteins in CSF has nothing to do with the
concentration of these in serum.
MAPA CONCEPTUAL
MAPA CONCEPTUAL
BIBLIOGRAFIA
MARTINEZ, L. Biología Molecular. ED UPB.
Segunda edición. Medellín. 2011.
HUANG Y-C, TSENG M-Y, et al. Increased
prothrombin, Apolipoprotein A-IV, and
Haptoglobin in the cerebrospinal fluid of
patients with Huntington`s disease. Enero 31,
2011